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Hydrophobic interaction chromatography correctly refolding proteins assisted by glycerol and urea gradients
Alternative TitleJ. Chromatogr. A
Li, JJ; Liu, YD; Wang, FW; Ma, GH; Su, ZG
2004-12-24
Source PublicationJOURNAL OF CHROMATOGRAPHY A
ISSN0021-9673
Volume1061Issue:2Pages:193-199
AbstractChromatographic columns packed with commercially available hydrophobic interaction chromatography (HIC) media were found to be able to suppress aggregation and nevertheless had a tendency to promote the structural misfolding resulting in higher soluble protein recovery and lower specific activity than that by dilution when they were used to refold lysozyme. a model protein. Moreover, this misfolding effect was exacerbated with increasing hydrophobicity of media. A novel strategy involving the combination of glycerol, a typical osmolyte, a urea gradient and commercially available HIC media was introduced to facilitate protein refolding correctly as well as improve mass recovery by providing a gradual change of the refolding environment in the HIC column. In this process, unfolded lysozyme was bound to Poros PE HIC column at high salt concentration and was released by a urea gradient followed by elution with refolding buffer in the presence of 50% (v/v) glycerol, resulting in 86.3% activity yield and 85% mass recovery with the refolded product of native specific activity For the absence of glycerol, only 50.9% activity yield and 59% specific activity recovery was obtained although mass recovery was closed to that in the presence of glycerol. It was also discovered that glycerol addition during elution process was necessary for correct refolding compared to mixing of glycerol with post-column fraction. The possible mechanism for refolding with this system vas proposed to be relevant to the formation of an on-pathway intermediate that could slowly reactivate. (C) 2004 Elsevier B.V. All rights reserved.; Chromatographic columns packed with commercially available hydrophobic interaction chromatography (HIC) media were found to be able to suppress aggregation and nevertheless had a tendency to promote the structural misfolding resulting in higher soluble protein recovery and lower specific activity than that by dilution when they were used to refold lysozyme. a model protein. Moreover, this misfolding effect was exacerbated with increasing hydrophobicity of media. A novel strategy involving the combination of glycerol, a typical osmolyte, a urea gradient and commercially available HIC media was introduced to facilitate protein refolding correctly as well as improve mass recovery by providing a gradual change of the refolding environment in the HIC column. In this process, unfolded lysozyme was bound to Poros PE HIC column at high salt concentration and was released by a urea gradient followed by elution with refolding buffer in the presence of 50% (v/v) glycerol, resulting in 86.3% activity yield and 85% mass recovery with the refolded product of native specific activity For the absence of glycerol, only 50.9% activity yield and 59% specific activity recovery was obtained although mass recovery was closed to that in the presence of glycerol. It was also discovered that glycerol addition during elution process was necessary for correct refolding compared to mixing of glycerol with post-column fraction. The possible mechanism for refolding with this system vas proposed to be relevant to the formation of an on-pathway intermediate that could slowly reactivate. (C) 2004 Elsevier B.V. All rights reserved.
KeywordRefolding Hydrophobic Interaction Chromatography Glycerol Lysozyme Gradient
SubtypeArticle
WOS HeadingsScience & Technology ; Life Sciences & Biomedicine ; Physical Sciences
DOI10.1016/j.chroma.2004.11.002
URL查看原文
Indexed BySCI
Language英语
WOS KeywordSIZE-EXCLUSION CHROMATOGRAPHY ; INCLUSION-BODIES ; ESCHERICHIA-COLI ; LYSOZYME ; RENATURATION ; AGGREGATION ; YIELD
WOS Research AreaBiochemistry & Molecular Biology ; Chemistry
WOS SubjectBiochemical Research Methods ; Chemistry, Analytical
WOS IDWOS:000225853400009
Citation statistics
Cited Times:33[WOS]   [WOS Record]     [Related Records in WOS]
Document Type期刊论文
Version出版稿
Identifierhttp://ir.ipe.ac.cn/handle/122111/4944
Collection研究所(批量导入)
Affiliation1.Chinese Acad Sci, Inst Proc Engn, Natl Key Lab Biochem Engn, Beijing 100080, Peoples R China
2.Dalian Univ Technol, Dalian 116024, LiaoNing, Peoples R China
3.Dalian Media Univ, Clin Coll 2, Dalian 116027, LiaoNing, Peoples R China
Recommended Citation
GB/T 7714
Li, JJ,Liu, YD,Wang, FW,et al. Hydrophobic interaction chromatography correctly refolding proteins assisted by glycerol and urea gradients[J]. JOURNAL OF CHROMATOGRAPHY A,2004,1061(2):193-199.
APA Li, JJ,Liu, YD,Wang, FW,Ma, GH,&Su, ZG.(2004).Hydrophobic interaction chromatography correctly refolding proteins assisted by glycerol and urea gradients.JOURNAL OF CHROMATOGRAPHY A,1061(2),193-199.
MLA Li, JJ,et al."Hydrophobic interaction chromatography correctly refolding proteins assisted by glycerol and urea gradients".JOURNAL OF CHROMATOGRAPHY A 1061.2(2004):193-199.
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